Our long term goal is to understand how nucleosomal structures affect DNA functions such as transcription and replication. Recently we discovered a new type of core histone, macroH2A (mH2A), which is present in about 3% of rat liver nucleosomes. It is three times the size of a conventional H2A, and has a novel hybrid structure consisting of a large nonhistone region, and a region that closely resembles a full length histone H2A. The nonhistone region contains a segment that resembles a leucine zipper, a structure involved in the dimerization of some proteins involved in transcriptional activation. These features suggest that nucleosomes that contain mH2A have novel functions, possibly involving interactions with other nuclear proteins. The goal of the research proposed in this application is to gain insights into these functions by investigating the following questions: -What effect does mH2A have on nucleosome structure? We will prepare nucleosomes that contain mH2A by immunopurification with an mH2A antibody, nucleosome reconstitution, or gel electrophoresis, and examine their histone composition by gel electrophoresis. We will examine the conformation and accessibility of their DNA by two methods: nuclease digestion and pyrimidine dimer formation. - Where is mH2A located in the chromatin? We will use immunofluorescence with an mH2A antibody to examine its distribution in nuclei and chromosomes. We will also examine the DNA sequences of nucleosomes immunopurified with an mH2A antibody; initially we will ask if sequences of transcriptionally active regions are preferentially present or absent. - What is the pattern of mH2A expression in different tissues, and during early development? We will purify chromatin from different tissues, and examine its mH2A content by gel electrophoresis. We will use immunofluorescence to examine the mH2A content of individual cells in tissues and developing embryos. We will also look for changes in mH2A expression during differentiation of cultured cells. - Does mH2A interact with itself or other nuclear proteins? We will look for proteins that interact with the nonhistone region of mH2A in vitro. We will also look for proteins that immunopurify with nucleosomes that contain mH2A and are absent from other nucleosomes. - What are the effects of altered mH2A expression? We will examine the effects of overexpression in cultured cells. We will also try to interfere with mH2A expression and function using antisense nucleic acids and dominant negative mutations. Results from these experiments should provide valuable clues to the function of this unusual histone. Understanding mH2A's function might give insights into human diseases such as cancer, which involve defects in gene regulation and replication.